The molecular mechanisms involved in DNA replication are being studied. Proteins involved in both phage lambda and E. coli replication are being purified and characterized in vitro. The present emphasis is to gain insight into the process of initiation of chromosome replication. The initiation of double-stranded Lambda dv plasmid DNA is being studied in vitro. This reaction requires two phage initiation proteins, O and P gene products, host proteins, RNA transcription in the region of the origin of replication and a specific site, ori, on the Lambda DNA. By analysis of in vitro constructed deletions of the ori region, I have found the minimal piece of DNA that functions in initiating DNA replication in vitro by and O and P dependent reaction. This piece of DNA is 95 base pairs and contains two O binding sites followed on the right by a region rich in adenine. I have also confirmed the domain structure of Lambda O suggested by genetic experiments. The DNA binding domain resides in the amino-terminal portion of O as shown by DNA protection experiments with the purified amino-terminal half of O protein. P protein most likely interacts with O protein through the carboxy-terminal portion of O since P protein forms as complex with intact O protein but not with the amino-terminal fragment of O.